Chia-Ling Chen

Blue light acts as a double-edged sword in regulating sexual development of Hypocrea jecorina (Trichoderma reesei).

The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1–2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.

Trichoderma reesei meiosis generates segmentally aneuploid progeny with higher xylanase- producing capability

BackgroundHypocrea jecorina is the sexual form of the industrial workhorse fungus Trichoderma reesei that secretes cellulases and hemicellulases to degrade lignocellulosic biomass into simple sugars, such as glucose and xylose. H. jecorina CBS999.97 is the only T. reesei wild isolate strain that is sexually competent in laboratory conditions. It undergoes a heterothallic reproductive cycle and generates CBS999.97(1-1) and CBS999.97(1-2) haploids with MAT1-1 and MAT1-2 mating-type loci, respectively. T. reesei QM6a and its derivatives (RUT-C30 and QM9414) all have a MAT1-2 mating type locus, but they are female sterile. Sexual crossing of CBS999.97(1-1) with either CBS999.97(1-2) or QM6a produces fruiting bodies containing asci with 16 linearly arranged ascospores (the sexual spores specific to ascomycetes). This sexual crossing approach has created new opportunities for these biotechnologically important fungi.ResultsThrough genetic and genomic analyses, we show that the 16 ascospores are generated via meiosis followed by two rounds of postmeiotic mitosis. We also found that the haploid genomes of CBS999.97(1-2) and QM6a are similar to that of the ancestral T. reesei strain, whereas the CBS999.97(1-1) haploid genome contains a reciprocal arrangement between two scaffolds of the CBS999.97(1-2) genome. Due to sequence heterozygosity, most 16-spore asci (>90%) contain four or eight inviable ascospores and an equal number of segmentally aneuploid (SAN) ascospores. The viable SAN progeny produced higher levels of xylanases and white conidia due to segmental duplication and deletion, respectively. Moreover, they readily lost the duplicated segment approximately two weeks after germination. With better lignocellulosic biomass degradation capability, these SAN progeny gain adaptive advantages to the natural environment, especially in the early phase of colonization.ConclusionsOur results have not only further elucidated T. reesei evolution and sexual development, but also provided new perspectives for improving T. reesei industrial strains.

The Tetraindole SK228 Reverses the Epithelial-to-Mesenchymal Transition of Breast Cancer Cells by Up-Regulating Members of the miR-200 Family

The results of recent studies have shown that metastasis, the most common malignancy and primary cause of mortality promoted by breast cancer in women, is associated with the epithelial-to-mesenchymal transition (EMT). The results of the current study show that SK228, a novel indole containing substance, exhibits anti-cancer activity. In addition, the effects of SK228 on the regulation of EMT in breast cancer cells as well as the underlying mechanism have been explored. SK228 was observed to induce a fibroblastoid to epithelial-like change in the appearance of various breast cancer cell lines and to suppress the migration and invasion of these cancer cells in vitro. Moreover, expression of E-cadherin was found to increase following SK228 treatment whereas ZEB1 expression was repressed. Expression of other major EMT inducers, including ZEB2, Slug and Twist1, is also repressed by SK228 as a consequence of up-regulation of members of the miR-200 family, especially miR-200c. The results of animal studies demonstrate that SK228 treatment leads to effective suppression of breast cancer growth and metastasis in vivo. The observations made in this investigation show that SK228 reverses the EMT process in breast cancer cells via an effect on the miR-200c/ZEB1/E-cadherin signalling pathway. In addition, the results of a detailed analysis of the in vivo anti-cancer activities of SK228, carried out using a breast cancer xenograft animal model, show that this substance is a potential chemotherapeutic agent for the treatment of breast cancer.

Anticancer efficacy of unique pyridine-based tetraindoles.

Results of previous studies demonstrated that the tetraindole, SK228, which has a high lipid but low water solubility, displayed moderate anticancer efficacy in a xenograft model of breast cancer. This finding led to the proposal that new, pyridine based tetraindole (PBT) analogs of SK228, containing tetraindole moieties distributed about central protonated pyridine cores, would have enhanced bioavailabilities and anticancer efficacies. Among the PBTs prepared and subjected to biological studies, 3f (FCW81) was observed to display the highest antiproliferative activity against the two triple negative breast cancer (TNBCs) cell lines, MDA-MB-231 and BT549. In addition, its mode of action was shown to involve G2/M arrest of the cell cycle along with the promotion of increased levels of cyclin B1 and p-chk2 and a decreased level of p-cdc2. DNA damage and induction of apoptosis caused by FCW81 was found to be associated with a decrease in DNA repair. Significantly, FCW81 displays therapeutic efficacy in a xenograft model of human breast cancer by not only serving to inhibit markedly the growth of cancer cells but also to block effectively cancer cell metastasis. Collectively, the results of these studies have led to the identification of novel pyridine-tetraindole based anticancer agents with potential use in TNBC therapy.

Aggregation-induced emission enhancement of anthracene-derived Schiff base compounds and their application as a sensor for bovine serum albumin and optical cell imaging

Three anthracene-based Schiff base complexes, R1-R3 (R1 = (E)-N´-((anthracen-10-yl)methylene)benzohydrazide; R2 = (E)-1-((anthracen-10-yl)methylene)-4-phenylsemicarbazide; and R3 = (E)-1-((anthracen-10-yl)methylene)-4-phenylthiosemicarbazide) were synthesized from 9-anthracenecarboxaldehyde, benzohydrazide, 4-phenylsemicarbazide and 4-phenylthiosemi-carbazide respectively, and characterized by various spectral techniques. The absorption spectral characteristics of R1-R3 were bathochromically tuned to the visible region by extending the π conjugation. These target compounds were weakly fluorescent in tetrahydrofuran (THF) solution because of rapid isomerization of the C=N double bond in the excited state. However, the aqueous dispersion of R1-R3 in the THF/water mixture by the gradual addition of water up to 90% resulted in an increase in the fluorescence intensity mainly due to aggregation-induced emission enhancement (AIEE) properties. The formation of nanoaggregates of R1-R3 were confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. The compounds R1-R3 are ideal probes for the fluorescence sensing of bovine serum albumin (BSA) and breast cancer cells by optical cell imaging.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels

ABSTRACT The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis. IMPORTANCE Trichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Hybrid Infertility: The Dilemma or Opportunity of Applying Sexual Development to Improve Trichoderma reesei Industrial Strains

Trichoderma reesei (Teleomorph Hypocrea jecorina) RUT-C30 and QM9414 strains are the principal industrial producers of enzymes that hydrolyze lignocellulosic biomass into simple sugars, such as glucose and xylose. These fungi were generated from the wild isolate QM6a via multiple rounds of random mutagenesis for enhanced cellulase production and/or catabolite derepression. Accumulating evidence indicates that their genomes have acquired multiple chromosomal alternations (including nucleotide mutations, segmental deletions and rearrangements) compared with that of QM6a. We found that RUT-C30 and QM9414, such as QM6a, efficiently mated with the H. jecorina CBS999.97 mating partner, including completed sexual development and meiosis. However, they generated more non-viable segmental aneuploidy (SAN) ascospores than the sexual crossing of CBS999.97 with QM6a. Our results indicate that extensive mutagenesis during strain improvements resulted in speciation, i.e., RUT-C30 and QM9414 are no longer the same species as QM6a and CBS999.97. Our finding is consistent with the classic chromosomal speciation model stating that chromosome rearrangements contribute to heterozygous hybrid infertility and serve as a genetic barrier between recently diverged species. We suggest that RUT-C30 and QM9414 are ideal models for deciphering molecular mechanisms by which new genetic barriers impeding reproduction arise. Hybridization between the industrial strains and the CBS999.97 wild isolates may generate SAN progeny that harbor existing or new beneficial phenotypes for economic applications.

Draft Genome Sequence of Burkholderia sp. Strain WAC0059, a Bacterium Isolated from the Medicinal Fungus Antrodia cinnamomea

Burkholderia sp. strain WAC0059 was isolated from a fruiting body of the medicinal fungus Antrodia cinnamomea collected in Taiwan. Here, we report the draft genome sequence of this bacterium to facilitate the investigation of its biology.

Nature-inspired design of tetraindoles: Optimization of the core structure and evaluation of structure-activity relationship.

Building on the initial successful optimization of a novel series of tetraindoles, a second generation of the compounds with changes in the core phenyl ring was synthesized to improve anticancer properties. 17 new compounds with different rigidity, planarity, symmetry and degree of conjugation of their core structures to 5-hydroxyindole units were synthesized. All the compounds were fully characterized and tested against breast cancer cell line (MDA-MB-231). The results revealed that the core structure is required for activity and it should be aromatic, rigid, planar, symmetrical and conjugated for optimal activity. Compound 29, which has strong anticancer activity against various tumor-derived cell lines, including Mahlavu (hepatocellular), SK-HEP-1 (hepatic), HCT116 (colon), MIA PaCa-2 (pancreatic), H441 (lung papillary), A549 (lung), H460 (non-small cell lung) and CL1-5 (lung carcinoma) with IC50 values ranging from 0.19 to 3.50μM, was generated after series of successive optimizations. It was found to induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in the non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice bearing xenografted MIA PaCa-2 human pancreatic cancer.